The present invention relates to isolated dirigent proteins and pinoresinol/lariciresinol reductases from Forsythia intermedia, Tsuga heterophylla and Thuja plicata, to nucleic acid sequences which code for dirigent proteins and pinoresinol/lariciresinol reductases from Forsythia intermedia, Tsuga heterophylla and Thuja plicata, and to vectors containing the sequences, host cells containing the sequences and methods of producing recombinant pinoresinol/lariciresinol reductases, recombinant dirigent protein and their mutants.
Lignans are a large, structurally diverse, class of vascular plant metabolites having a wide range of physiological functions and pharmacologically important properties (Ayres, D. C., and Loike, J. D. in Chemistry and Pharmacology of Natural Products. Lignans. Chemical, Biological and Clinical Properties, Cambridge University Press, Cambridge, England (1990); Lewis et al., in Chemistry of the Amazon, Biodiversity Natural Products, and Environmental Issues, 588, (P. R. Seidl, O. R. Gottlieb and M. A. C. Kaplan) 135-167, ACS Symposium Series, Washington D.C. (1995)). Because of their pronounced antibiotic properties (Markkanen, T. et al., Drugs Exptl. Clin. Res. 7:711-718 (1981)), antioxidant properties (Faurxc3xa9, M. et al., Phytochemistry 29:3773-3775 (1990); Osawa, T. et al., Agric. Biol. Chem. 49:3351-3352 (1985)) and antifeedant properties (Harmatha, J., and Nawrot, J., Biochem. Syst. Ecol. 12:95-98 (1984)), a major role of lignans in vascular plants is to help confer resistance against various opportunistic biological pathogens and predators. Lignans have also been proposed as cytokinins (Binns, A. N. et al., Proc. Natl. Acad. Sci. USA 84:980-984 (1987)) and as intermediates in lignification (Rahman, M. M. A. et al., Phytochemistry 29:1861-1866 (1990)), suggesting a critical role in plant growth and development. It is widely held that elaboration of biochemical pathways to lignins/lignans and related substances from phenylalanine (tyrosine) was essential for the successful transition of aquatic plants to their vascular dry-land counterparts (Lewis, N. G., and Davin, L. B., in Isoprenoids and Other Natural Products. Evolution and Function, 562 (W. D. Nes, ed) 202-246, ACS Symposium Series: Washington, D.C. (1994)), some four hundred and eighty million years ago (Graham, L. E., Origin of Land Plants, John Wiley and Sons, Inc., New York, N.Y. (1993)).
Based on existing chemotaxonomic data, lignans are present in xe2x80x9cprimitivexe2x80x9d plants, such as the fern Blechnum orientate (Wada, H. et al., Chem. Pharm. Bull. 40:2099-2101 (1992)) and the hornworts, e.g., Dendroceros japonicus and Megaceros flagellaris (Takeda, R. et al., in Bryophytes. Their Chemistry and Chemical Taxonomy, Vol. 29 (Zinsmeister, H. D. and Mues, R. eds) pp. 201-207, Oxford University Press: New York, N.Y. (1990); Takeda, R. et al., Tetrahedron Lett. 31:4159-4162 (1990)), with the latter recently being classified as originating in the Silurian period (Graham, L. E., J. Plant Res. 109: 241-252 (1996)). Interestingly, evolution of both gymnosperms and angiosperms was accompanied by major changes in the structural complexity and oxidative modifications of the lignans (Lewis, N. G., and Davin, L. B., in Isoprenoids and Other Natural Products. Evolution and Function, 562 (W. D. Nes, ed) 202-246, ACS Symposium Series: Washington, D.C. (1994); Gottlieb, O. R., and Yoshida, M., in Natural Products of Woody Plants. Chemicals Extraneous to the Lignocellulosic Cell Wall (Rowe, J. W. and Kirk, C. H. eds) pp. 439-511, Springer Verlag: Berlin (1989)). Indeed, in some species, such as Western Red Cedar (Thuja plicata), lignans can contribute extensively to heartwood formation/generation by enhancing the resulting heartwood color, quality, fragrance and durability.
In addition to their functions in plants, lignans also have important pharmacological roles. For example, podophyllotoxin, as its etoposide and teniposide derivatives, is an example of a plant compound that has been successfully employed as an anticancer agent (Ayres, D. C., and Loike, J. D. in Chemistry and Pharmacology of Natural Products. Lignans. Chemical, Biological and Clinical Properties, Cambridge University Press, Cambridge, England (1990)). Antiviral properties have also been reported for selected lignans. For example, (xe2x88x92)-arctigenin (Schrxc3x6der, H. C. et al., Z. Naturforsch. 45c, 1215-1221 (1990)), (xe2x88x92)-trachelogenin (Schrxc3x6der, H. C. et al., Z. Naturforsch. 45c, 1215-1221 (1990)) and nordihydroguaiaretic acid (Gnabre, J. N. et al., Proc. Natl. Acad. Sci. USA 92:11239-11243 (1995)) are each effective against HIV due to their pronounced reverse transcriptase inhibitory activities. Some lignans, e.g., matairesinol (Nikaido, T. et al., Chem. Pharm. Bull. 29:3586-3592 (1981)), inhibit cAMP-phosphodiesterase, whereas others enhance cardiovascular activity, e.g., syringaresinol xcex2-D-glucoside (Nishibe, S. et al., Chem. Pharm. Bull. 38:1763-1765 (1990)). There is also a high correlation between the presence, in the diet, of the xe2x80x9cmammalianxe2x80x9d lignans or xe2x80x9cphytoestrogensxe2x80x9d, enterolactone and enterodiol, formed following digestion of high fiber diets, and reduced incidence rates of breast and prostate cancers (so-called chemoprevention) (Axelson, M., and Setchell, K. D. R., FEBS Lett. 123:337-342 (1981); Adlercreutz et al., J. Steroid Biochem. Molec. Biol. 41:3-8 (1992); Adlercreutz et al., J. Steroid Biochem. Molec. Biol. 52:97-103 (1995)). The xe2x80x9cmammalian lignans,xe2x80x9d in turn, are considered to be derived from lignans such as matairesinol and secoisolariciresinol (Boriello et al., J. Applied Bacteriol., 58:3743 (1985)).
The biosynthetic pathways to the lignans are only now being defined, although there are no prior art reports of the isolation of enzymes or genes involved in the lignan biosynthetic pathway. Based on radiolabeling experiments with crude enzyme extracts from Forsythia intermedia, it was first established that entry into the 8,8xe2x80x2-linked lignans, which represent the most prevalent dilignol linkage known (Davin, L. B., and Lewis, N. G., in Rec. Adv. Phytochemistry, Vol. 26 (Stafford, H. A., and Ibrahim, R. K., eds), pp. 325-375, Plenum Press, New York, N.Y. (1992)), occurs via stereoselective coupling of two achiral coniferyl alcohol molecules, in the form of oxygenated free radicals, to afford the furofuran lignan (+)-pinoresinol (Davin, L. B., Bedgar, D. L., Katayama, T., and Lewis, N. G., Phytochemistry 31:3869-3874 (1992); Parxc3xa9, P. W. et al., Tetrahedron Lett. 35:47314734 (1994)) (FIG. 1).
Bimolecular phenoxy radical coupling reactions, such as the stereoselective coupling of two achiral coniferyl alcohol molecules to afford the furofuran lignan (+)-pinoresinol, are involved in numerous biological processes. These are presumed to include lignin formation in vascular plants (M. Nose et al., Phytochemistry 39:71 (1995)), lignan formation in vascular plants (N. G. Lewis and L. B. Davin, ACS Symp. Ser. 562:202 (1994); P. W. Parxc3xa9 et al., Tetrahedron Lett. 35:4731 (1994)), suberin formation in vascular plants (M. A. Bernards et al., J. Biol. Chem. 270:7382 (1995)), fruiting body development in fungi (J. D. Bu""Lock et al., J. Chem. Soc. 2085 (1962)), insect cuticle melanization and sclerotization (M. Miessner et al., Helv. Chim. Acta 74:1205 (1991); V. J. Marmaras et al., Arch. Insect Biochem. Physiol. 31:119 (1996)), the formation of aphid pigments (D. W. Cameron and Lord Todd, in Organic Substances of Natural Origin. Oxidative Coupling of Phenols, W. I. Taylor and A. R. Battersby, Eds. (Dekker, New York, 1967), Vol. 1, p.203), and the formation of algal cell wall polymers (M. A. Ragan, Phytochemistry 23:2029 (1984)).
In contrast to the marked regiochemical and/or stereochemical specificities observed in the biosynthesis of the foregoing lignin and lignan substances in vivo, all previously described chemical (J. Iqbal et al., Chem. Rev. 94:519 (1994)) and enzymatic (K. Freudenberg, Science 148:595 (1965)) bimolecular phenoxy radical coupling reactions in vitro have lacked strict regio- and stereospecific control. That is, if chiral centers are introduced during coupling in vitro, the products are racemic, and different regiochemistries can result if more than one potential coupling site is present. Thus, the ability to generate a particular enantiomeric form or a specific coupling product in vitro is not under explicit control. Consequently, it is inferred that a mechanism exists in vivo to control the regiochemistry and stereochemistry of bimolecular phenoxy radical coupling reactions leading to the formation of, for example, lignans.
In Forsythia intermedia, and presumably other species, (+)-pinoresinol, the product of the stereospecific coupling of two E-coniferyl alcohol molecules, undergoes sequential reduction to generate (+)-lariciresinol and then (xe2x88x92)-secoisolariciresinol (Katayama, T. et al., Phytochemistry 32:581-591 (1993); Chu, A. et al., J. Biol. Chem. 268:27026-27033 (1993)) (FIG. 1). While it has hitherto been unclear whether more than one reductase is required to catalyze the sequential steps, the reductions proceed via abstraction of the pro-R hydride of NADPH, resulting in an xe2x80x9cinversionxe2x80x9d of configuration at both the C-7 and C-7xe2x80x2 positions of the products, (+)-lariciresinol and (xe2x88x92)-secoisolariciresinol (Chu, A., et al., J. Biol. Chem. 268:27026-27033 (1993)). (xe2x88x92)-Matairesinol is subsequently formed via dehydrogenation of (xe2x88x92)-secoisolariciresinol, further metabolism of which presumably affords lignans such as the antiviral (xe2x88x92)-trachelogenin in Ipomoea cairica and (xe2x88x92)-podophyllotoxin in Podophyllum peltatum. 
Thus, the stereospecific formation of (+)-pinoresinol and the subsequent reductive steps giving (+)-lariciresinol and (xe2x88x92)-secoisolariciresinol are pivotal points in lignan metabolism, since they represent entry into the furano, dibenzylbutane, dibenzylbutyrolactone and aryltetrahydronaphthalene lignan subclasses. Additionally, it should be noted that while lignans are normally optically active, the particular enantiomer present may differ between plant species. For example, (xe2x88x92)-pinoresinol occurs in Xanthoxylum ailanthoides (Ishii et al., Yakugaku Zasshi, 103:279-292 (1983)), and (xe2x88x92)-lariciresinol is present in Daphne tangutica (Lin-Gen, et al., Planta Medica, 45:172-176 (1982)). The optical activity of a particular lignan may have important ramifications regarding biological activity. For example, (xe2x88x92)-trachelogenin inhibits the in vitro replication of HW-1, whereas its (+)-enantiomer is much less effective (Schroder et al., Naturforsch. 45c: 1215-1221(1990)).
In accordance with the foregoing, in one aspect of the invention it has now been discovered that a 78-kD dirigent protein is involved in conferring stereospecificity in 8,8xe2x80x2-linked lignan formation. This protein has no detectable catalytically active oxidative center and apparently serves only to bind and orient coniferyl alcohol-derived free radicals, which then undergo stereoselective coupling to form (+)-pinoresinol. The formation of free-radicals, in the first instance, requires the oxidative capacity of either a nonspecific oxidase or even a non-enzymatic electron oxidant. In another aspect of the invention, it has been discovered that a single enzyme, designated pinoresinol/lariciresinol reductase, catalyzes the conversion of pinoresinol to lariciresinol and then to secoisolariciresinol. Thus, one aspect of the invention relates to isolated dirigent proteins and to isolated pinoresinol/lariciresinol reductases, such as, for example, those from Forsythia intermedia, Thuja plicata and Tsuga heterophylla. 
In other aspects of the invention, cDNAs encoding dirigent protein from several plant species have been isolated and sequenced, and the corresponding amino acid sequences have been deduced. Also, cDNAs encoding pinoresinol/lariciresinol reductase from several plant species have been isolated and sequenced, and the corresponding amino acid sequences have been deduced.
Thus, the present invention relates to isolated proteins and to isolated DNA sequences which code for the expression of dirigent protein or pinoresinol/lariciresinol reductase. In other aspects, the present invention is directed to replicable vectors comprising a nucleic acid sequence which codes for a pinoresinol/lariciresinol reductase or for a dirigent protein. The present invention is also directed to a base sequence sufficiently complementary to at least a portion of a pinoresinol/lariciresinol reductase DNA or RNA, or to at least a portion of a dirigent protein DNA or RNA, to enable hybridization therewith. The aforesaid complementary base sequences include, but are not limited to: antisense pinoresinol/lariciresinol reductase RNA; antisense dirigent protein RNA; fragments of DNA that are complementary to a pinoresinovlariciresinol reductase DNA, or to a dirigent protein DNA, and which are therefore useful as polymerase chain reaction primers, or as probes for pinoresinol/lariciresinol reductase genes, dirigent protein genes, or related genes.
In yet other aspects of the invention, modified host cells are provided that have been transformed, transfected, infected and/or injected with a replicable vector and/or DNA sequence of the invention. Thus, the present invention provides for the recombinant expression of pinoresinol/lariciresinol reductases and dirigent proteins in plants, animals, microbes and in cell cultures. The inventive concepts described herein may be used to facilitate the production, isolation and purification of significant quantities of recombinant pinoresinol/lariciresinol reductase or dirigent protein, or of their enzyme products, in plants, animals, microbes or cell cultures.